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The analysis of mutations and exon deletions at TSC2 gene in angiomyolipomas associated with tuberous sclerosis complex
Experimental and Molecular Pathology 97 (2014) 440–444
Contents lists available at ScienceDirect
Experimental and Molecular Pathology journal homepage: www.elsevier.com/locate/yexmp
The analysis of mutations and exon deletions at TSC2 gene in angiomyolipomas associated with tuberous sclerosis complex Heung-Mo Yang a,1, Hye-Jung Choi a,1, Doo-Pyo Hong a, Sung-Yeon Joo a,b, Na-Eun Lee a,b, Ji-Young Song c, Yoon-La Choi d,i, Jeeyun Lee e,i, Dongil Choi f,i, BoKyung Kim g,i, Hyo-Jun Park a,h, Jae-Berm Park a,h,⁎, Sung Joo Kim a,h,i,⁎ a
Transplantation Research Center, Samsung Biomedical Research Institute, Samsung Medical Center, Seoul, Republic of Korea Samsung Advanced Institute for Health Sciences & Technology, Graduate School, Department of Health Sciences & Technology, Sungkyunkwan University, Republic of Korea c Laboratory of Cancer Genomics and Molecular Pathology, Samsung Biomedical Research Institute, Samsung Medical Center, Seoul, Republic of Korea d Department of Pathology, Samsung Medical Center, Samsung Biomedical Research Institute, Sungkyunkwan University School of Medicine, Seoul, Republic of Korea e Department of Hematological oncology, Samsung Medical Center, Samsung Biomedical Research Institute, Sungkyunkwan University School of Medicine, Seoul, Republic of Korea f Department of Radiology, Samsung Medical Center, Samsung Biomedical Research Institute, Sungkyunkwan University School of Medicine, Seoul, Republic of Korea g Department of Radiation Oncology, Samsung Medical Center, Samsung Biomedical Research Institute, Sungkyunkwan University School of Medicine, Seoul, Republic of Korea h Department of Surgery, Samsung Medical Center, Samsung Biomedical Research Institute, Sungkyunkwan University School of Medicine, Seoul, Republic of Korea i Sarcoma Research Center, Samsung Medical Center, Seoul, Republic of Korea b
a r t i c l e
i n f o
Article history: Received 2 September 2014 Accepted 12 September 2014 Available online 1 October 2014 Keywords: Angiomyolipoma Tuberous sclerosis complex TSC2 gene Mutation Exon deletion
a b s t r a c t Angiomyolipomas (AMLs) are relatively rare hamartomatous or benign tumors that occasionally occur as part of tuberous sclerosis complex (TSC). Mutations in either of the two genes, TSC1 and TSC2, have been attributed to the development of TSC. Between 1994 and January 2009, 83 patients were diagnosed with AML at the Samsung Medical Center. In that group of patients, 5 (6%) had AML with TSC (AML-TSC). Mutational analysis of the TSC2 gene was performed using 7 samples from the 5 AML-TSC patients and 14 samples from 14 patients with sporadic AML without TSC (AML-non-TSC). From this analysis, mutations in TSC genes were identified in 5 samples from the AMLTSC patients (mutation detection rate = 71%) and 3 samples from AML-non-TSC patients (mutation detection rate = 21%). In the case of AML-TSC, 6 mutations were found including 3 recurrent mutations and 3 novel mutations, while in the case of AML-non-TSC, 4 mutations were identified once, including 1 novel mutation. Also MLPA analysis of the TSC2 gene showed that TSC2 exon deletion is more frequently observed in AML-TSC patients than in AML-non-TSC patients. This is the first mutation and multiplex ligation-dependent probe amplification (MLPA) analyses of TSC2 in Korean AMLs that focus on TSC. This study provides data that are representative of the distribution of mutations and exon deletions at TSC genes in clinically diagnosed AML-TSC cases of the Korean population. © 2014 Elsevier Inc. All rights reserved.
1. Introduction Angiomyolipomas (AMLs) are relatively rare hamartomatous or benign tumor-like lesions composed of three elements: blood vessels, smooth muscles, and adipose tissues (Folpe and Kwiatkowski, 2010). Classical AML possesses the three mentioned elements but rarely contains oncocytic, cystic, or epithelioid morphologic characters; in contrast, such features are present in atypical and epithelioid AML
⁎ Corresponding authors at: Department of Surgery, Samsung Medical Center, Sungkyunkwan University School of Medicine, 50 Irwon-Dong, Gangnam-Gu, Seoul 135-710, Republic of Korea. E-mail addresses: [email protected], [email protected] (J.-B. Park), [email protected], [email protected] (S.J. Kim). 1 These authors contributed equally to this work as first and corresponding authors.
http://dx.doi.org/10.1016/j.yexmp.2014.09.013 0014-4800/© 2014 Elsevier Inc. All rights reserved.
(Belanger et al., 2005). In epithelioid AML, malignant behavior has been reported with local recurrence and distant metastasis, while classic AML is benign (Cibas et al., 2001; Kawaguchi et al., 2002). According to previous studies, AML primarily occurs in the kidney but can occur in the liver and other organs (Eble, 1998). Although the majority of AMLs are sporadic, in 20% of AML patients, they manifest with tuberous sclerosis complex (TSC); additionally, 60–80% of adult renal AML patients have TSC (Eble, 1998; Rakowski et al., 2006). TSC is an autosomal dominant disorder that is characterized by the development of multiple hamartomas in many internal organs such as the brain, heart, lung, skin, and kidney (Roach et al., 1998). Wellknown clinical manifestations of TSC include epilepsy, learning difficulties, behavioral problems, and skin lesions. The genetic basis of TSC comes from the mutation of either of the two unlinked genes, TSC1 and TSC2 (Povey et al., 1994). The human TSC1 gene is located on
H.-M. Yang et al. / Experimental and Molecular Pathology 97 (2014) 440–444
chromosome 9q34 and consists of 23 exons; the corresponding mRNA transcript is 8.6 kb, with a 3.5 kb coding region encoding a 130-kDa protein comprising 1164 amino acids (van Slegtenhorst et al., 1997). The TSC2 gene is located on chromosome 16p13.3 and contains 41 exons encoding a 200-kDa protein of 1807 amino acids (European Chromosome 16 Tuberous Sclerosis, 1993; Povey et al., 1994). The mutational spectra of the TSC genes are very heterogeneous, and no hotspots for mutations have been reported. In this study, we analyzed mutations and exon deletions of TSC2 gene in AML patients with the goal of determining the mutation in Korean individuals with both AML and TSC and providing information on the genetic character of tuberous sclerosis complex. 2. Materials and methods 2.1. Patients Between 1994 and January 2009, 83 patients were treated surgically for pathology-diagnosed AML at the Samsung Medical Center. Clinical information was collected retrospectively from patient medical records, including age at presentation, gender, involved organ, size, multiplicity, disease recurrence, and follow-up information. Clinicians assessed the general clinical features of TSC patients in accordance with the TSC diagnosis criteria set forth by the Tuberous Sclerosis Consensus Conference (Roach et al., 1998). TSC diagnosis was categorized as definite in patients with AML and other major symptoms of TSC, such as facial angiofibromas or lymphangiomyomatosis, and TSC diagnosis was categorized as probable in patients with multiple large AMLs or AML with 1 minor feature such as multiple renal cysts or liver lesions. For the purpose of mutational analysis, we divided the AML patients into two groups according to the presence (AML-TSC; n = 5) or absence (AMLnon-TSC; n = 14) of an association with TSC (Fig. 1).
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using TSC2 genomic sequence information (GenBank accession numbers AC005600.1, NM_000548.3, and NP_000539.2). PCR was performed using h-Taq DNA polymerase (Solgent, Daejeon, Korea) in 30-μl reaction volumes. Reactions were denatured at 95 °C for 20 s, annealed at 60 °C for 40 s, and extended at 72 °C for 1 min. Following the PCR reactions, we performed DNA sequencing on an ABI 3730XL DNA sequencer using a Big Dye Terminator v3.1 Cycle Sequencing Kit (Applied Biosystems, Foster City, CA, USA). 2.3. Multiplex ligation-dependent probe amplification (MLPA) analysis Genomic DNA was prepared from paraffin-embedded AML-TSC and AML-non-TSC patient tissues. Paraffin embedded human lung tissues (n = 4) were used as normal control samples. Probable identified deletions were confirmed using an MLPA kit (SALSA MLPA P046-C1 TSC2, Lot 1011; MRC-Holland, Amsterdam, The Netherlands), which contains probes for 41 TSC2 exons, according to the manufacturer's instructions. Reaction products were separated by capillary electrophoresis on an ABI 3700 sequencer (Applied Biosystems, Foster City, CA, USA) and data were analyzed using Genemarker software, version 2.5.2 (Softgenetics, State College, PA, USA). This program identifies peaks as ‘normal’ when the ratio of sample peak height or area to that of control samples falls between 0.75 and 1.30, ‘deleted’ when the ratio is b0.75, and ‘duplicated’ when the ratio is N1.30. 2.4. Statistical analysis Statistical comparisons were performed using Student's t-test with IBM SPSS (IBM Co., Armonk, NY, USA) computer software (version 12.0). Results were considered to be significant when p-values were less than 0.05.
2.2. Mutational analysis
3. Results
The mutational analysis of TSC2 genes was performed using tissue samples (n = 7) from AML-TSC patients (n = 5), including recurrent cases, and tissue samples (n = 14) from selected AML-non-TSC patients (n = 14). Genomic DNA extract kits (QIAamp® DNA FFPE Tissue Kit; QIAGEN, Valencia, CA, USA) were used according to the manufacturer's recommendations. TSC2 primers (Supplemental Table 2) were designed
3.1. Patient characteristics The clinical characteristics of patients with AML-TSC (n = 5) and AML-non-TSC (n = 14) are described in Table 1. Five of the 83 patients (6%) were AML-TSC patients; among these patients, 3 fulfilled the criteria for definitive tuberous sclerosis, and 2 were considered probable for TSC (Table 1). Comparing the clinical features between groups, patients with AML-TSC had significantly larger average AML tissue diameter(s) compared to those of the AML-non-TSC (21.4 cm vs. 5.2 cm, P b 0.001) and had multiple lesions more often than did the AML group (P = 0.004, Supplemental Table 1). Although the difference was not statistically significant, the AML-TSC group was younger and had a higher percentage of women than the AML-non-TSC group. Furthermore, we identified 3 cases of disease recurrence among all AML patients: 2 cases in the AML-TSC group and 1 case in the AMLnon-TSC group. 3.2. Mutations of TSC2 gene in AML-TSC patients
Fig. 1. Schematic diagram of TSC2 gene mutation and MLPA analysis in AML patients. For the mutation and MLPA analysis, the AML patients were divided into two groups according to the presence (AML-TSC; n = 5) or absence (AML-non-TSC; n = 14) of an association with TSC.
We performed a mutational analysis of the TSC2 coding exons in 7 samples from 5 AML-TSC patients. From the mutational analysis of the AML-TSC group samples, TSC2 gene mutations were detected in 5 samples (Patients Nos. 19A/B, 54A/B and 79), resulting in a 71% mutation detection rate (Table 1, Fig. 2A–F). As shown in Table 1, a total of 9 mutations were detected in the TSC2 genes of 3 patients from the AML-TSC group. We identified 3 unique mutations that were only detected in samples from patients with recurrent disease. Of the 6 types of mutations identified in the TSC2 gene, 1 of the 6 mutations was a nonsense mutation that resulted in an early termination codon, and the other 5 were silent mutations that altered the nucleotide sequence without affecting the TSC2 amino acid sequence (Table 1).
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Table 1 Clinical features and TSC2 gene mutation analysis of AML-TSC and AML-non-TSC patients.
3.3. Mutations in the TSC2 gene in AML-non-TSC patients From the 78 AML-non-TSC patients, we selected 14 patient samples to compare with the samples from the AML-TSC patients. Although only 1 patient with multiple tumors (a minor TSC symptom) is represented in the 14 patient samples, we could not find any other symptoms for TSC diagnosis in the 14 non-TSC patients. A mutational analysis of the TSC2 gene was performed for the 14 samples from the 14 AML-non-TSC patients. From the results of our mutational analysis, we identified 4 mutations in 3 of the 14 AML patients, representing a mutation detection rate of 21%. The 4 TSC2 mutations included 2 missense mutations, i.e., the c.1378 GNA mutation in exon 13 causing p.A460T and the c.1100 GNA mutation in exon 10 causing p.R367Q, and 2 nonsense mutations. Three of these mutations had been previously reported, while 1 mutation (c.1556_1557insG in exon 14) had not been reported (Table 1, Fig. 2G–J).
3.4. MLPA analysis of the TSC2 gene in AML-TSC and AML-non-TSC patients After mutational analysis, we analyzed exon deletion in TSC2 gene using MLPA analysis with 7 samples from 5 AML-TSC patients and 11 samples from 11 AML-non-TSC patients. According to the MLPA analysis, 4 to 8 exon deletions were identified in 7 samples of AML-TSC patients (average 5.9 deletion events/sample). Hence, in 11 samples of AMLnon-TSC patients, 0 to 5 exon deletions were identified (average 3.3 deletion events/sample; Table 1, Supplementary Fig. 1). In addition, deletions
in the TSC2 gene are commonly detected in exons 03, 04, 40 and 41 in AML patients (Supplementary Fig. 2) and TSC2 exon deletion is observed more frequently in AML-TSC patients than in AML-non-TSC patients. 4. Discussion Angiomyolipomas are benign tumors that develop mostly in the kidney and contain varying amounts of smooth muscle, fat, and blood vessels. AML is a relatively rare neoplasm that has been identified in less than 0.2% of the general population, although it is extremely common in patients with tuberous sclerosis complex (Folpe and Kwiatkowski, 2010; Lane et al., 2008). Among TSC patients, AMLs are an important diagnostic criteria in both adults and children (Jozwiak et al., 2000), and the AMLs ultimately leading to end-stage renal failure and renal disease as causes of death (Shepherd et al., 1991). Moreover, mutations in the TSC1 and TSC2 genes have been attributed to the development of TSC, and mutational analysis is used in the diagnosis of TSC. In this study, we reported the clinical features of AML patients and firstly performed a mutational analysis of the coding exons of the TSC2 gene to investigate the association of mutations with TSC in Korean populations. TSC2, rather than TSC1, was studied because TSC1 gene mutations are more rare than TSC2 gene mutations (Qin et al., 2011) and because we were unable to detect TSC1 mutations in 7 samples from 5 AML-TSC patients (data not shown). Our study demonstrated a relatively low prevalence rate for AML associated with TSC (5 of 83 patients, 6%) compared with the rate of 20%
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Fig. 2. Representative sequencing traces demonstrating TSC2 mutations. A–F. Mutations in the TSC2 genes of AML-TSC patients. G–J. Mutations in the TSC2 genes of AML-non-TSC patients. Red arrows indicate the point of mutation.
reported by Lendvay and Marshall (2003). However, our identified rate is consistent with previous studies finding that fewer than 10% of all AMLs occur in individuals with TSC (Aydin et al., 2009; Chen et al., 1997; Tong et al., 1990). According to previous reports, AMLs associated with TSC were found in significantly younger patients, had a larger mean tumor size, and had more lesions than did those without TSC (Aydin et al., 2009; Nelson and Sanda, 2002). Additionally, AML is approximately twice as frequent in women as in men, both in the general population and in TSC patients (Shepherd et al., 1991; Tong et al., 1990). Similarly, our study demonstrated that AML-TSC patients had significantly larger AMLs, multiple lesions, and frequent disease recurrences (Table 1, Supplemental Table 1). In TSC patients, mutations in the TSC1 and TSC2 genes are detected in a maximum of 80–90% of patients, with only a single relatively small series achieving a mutation detection rate of 91% (Jansen et al., 2008). In this study, we identified 5 mutations in 7 samples from the AML-TSC group, corresponding to a mutation detection rate of 71%, and all of these mutations were in the TSC2 gene. The mutation rate observed in our samples is lower than that of other western countries and Japan (Dabora et al., 2001; Jones et al., 1997, 1999; Yamashita et al., 2000). Moreover, many reports have suggested that mutations in TSC2 are about five times more common in the sporadic TSC population than mutations in TSC1. Although the incidence of mutations in our study is slightly lower than in previous reports, we observed a lower mutation rate in the TSC2 gene (21%) compared to that found for the AML-TSC group (Table 1). To date, over 1500 different mutations in TSC1 and TSC2 have been reported (Cheadle et al., 2000). Among these mutations, deletions, nonsense, and missense mutations occur at nearly equal frequencies in TSC2 (22–27%). According to a previous study, most lesions are distributed over the entire TSC2 gene region and include point mutations leading to nonsense mutations, small deletions and insertions, splice site changes, and missense mutations (Napolioni and Curatolo, 2008). In our MLPA assay, we observed several deleted and amplified TSC2 exons
(Supplemental Fig. 1). TSC2 exon deletion was observed more frequently in the samples of AML-TSC patients (average 5.9 events) than in the samples of AML-non-TSC patients (average 3.3 events). In addition, not only mutation presence and deletion presence in a specific gene are important, but also mutation status and genomic rearrangement are necessary factors to understand genetic alteration. Therefore, the patterns of the gene inactivation (one/two copy gene inactivations) and genomic event (inversion, frame-shift, insertion) are valuable information. We also know that LOH analysis is needed for the analysis of genomic alteration as well as for the inactivation pattern of a specific gene. However, unfortunately, it is difficult to perform LOH analysis, because we have no normal part of tissue sample from patients. Based on our analysis, it seems that a relatively high rate of mutation and deletion events in the TSC2 gene might be of concern for TSC progression and diagnosis. In our results, there are two pairs of samples from the same patients (patient nos. 19A/B and 54A/B). It seems that the mutations occurred independently, with the exception of mutation of c.228 CNT in exon 03. This mutation has a possibility of being a germline mutation because it was detected in both samples from one patient (patient no. 54A/B); however, we cannot make any assertions in this matter. In conclusion, our study is the first analysis identifying TSC2 gene mutations and deletions from AML-TSC patients in Korea. Among 83 AML patients, 5 patients were designated as having AML-TSC. The results of our mutational analysis of TSC2 genes in AML-TSC show that six different mutations were identified (a mutation detection rate of 71%), while 4 different mutations were identified in AML-non-TSC (a mutation detection rate of 21%). Moreover, TSC2 exon deletion is observed more frequently in AML-TSC patients than in AML-non-TSC patients. Although the incidence of TSC mutations is slightly lower than that found in previous mutational analyses, TSC mutation-related clinical features of AML-TSC patients were similar to previous reports. Moreover, for recurrent AML patients, repeated mutations of TSC2 were observed in multiple unrelated patients and in multiple samples from
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the same patient. The identification of additional TSC2 gene mutations and exon deletions will help confirm a TSC diagnosis and will aid in the clinical management of AML-TSC patients. Supplementary data to this article can be found online at http://dx. doi.org/10.1016/j.yexmp.2014.09.013. Conflict of interest statement No potential conflicts of interest were disclosed. Acknowledgment This research was supported by Samsung Biomedical Research Institute grant, SBRI C-B1-105-3 and supported by Basic Science Research Program through the National Research Foundation of Korea (NRF) funded by the Ministry of Education (NRF-2013R1A1A2063324). References Aydin, H., et al., 2009. Renal angiomyolipoma: clinicopathologic study of 194 cases with emphasis on the epithelioid histology and tuberous sclerosis association. Am. J. Surg. Pathol. 33, 289–297. Belanger, E.C., et al., 2005. Epithelioid angiomyolipoma of the kidney mimicking renal sarcoma. Histopathology 47, 433–435. Cheadle, J.P., et al., 2000. Molecular genetic advances in tuberous sclerosis. Hum. Genet. 107, 97–114. Chen, S.S., et al., 1997. Renal angiomyolipoma—experience of 20 years in Taiwan. Eur. Urol. 32, 175–178. Cibas, E.S., et al., 2001. Malignant epithelioid angiomyolipoma (‘sarcoma ex angiomyolipoma’) of the kidney: a case report and review of the literature. Am. J. Surg. Pathol. 25, 121–126. Dabora, S.L., et al., 2001. Mutational analysis in a cohort of 224 tuberous sclerosis patients indicates increased severity of TSC2, compared with TSC1, disease in multiple organs. Am. J. Hum. Genet. 68, 64–80. Eble, J.N., 1998. Angiomyolipoma of kidney. Semin. Diagn. Pathol. 15, 21–40. European Chromosome 16 Tuberous Sclerosis, C., 1993. Identification and characterization of the tuberous sclerosis gene on chromosome 16. Cell 75, 1305–1315.
Folpe, A.L., Kwiatkowski, D.J., 2010. Perivascular epithelioid cell neoplasms: pathology and pathogenesis. Hum. Pathol. 41, 1–15. Jansen, F.E., et al., 2008. Overlapping neurologic and cognitive phenotypes in patients with TSC1 or TSC2 mutations. Neurology 70, 908–915. Jones, A.C., et al., 1997. Molecular genetic and phenotypic analysis reveals differences between TSC1 and TSC2 associated familial and sporadic tuberous sclerosis. Hum. Mol. Genet. 6, 2155–2161. Jones, A.C., et al., 1999. Comprehensive mutation analysis of TSC1 and TSC2-and phenotypic correlations in 150 families with tuberous sclerosis. Am. J. Hum. Genet. 64, 1305–1315. Jozwiak, S., et al., 2000. Usefulness of diagnostic criteria of tuberous sclerosis complex in pediatric patients. J. Child Neurol. 15, 652–659. Kawaguchi, K., et al., 2002. Malignant transformation of renal angiomyolipoma: a case report. Am. J. Surg. Pathol. 26, 523–529. Lane, B.R., et al., 2008. Clinical correlates of renal angiomyolipoma subtypes in 209 patients: classic, fat poor, tuberous sclerosis associated and epithelioid. J. Urol. 180, 836–843. Lendvay, T.S., Marshall, F.F., 2003. The tuberous sclerosis complex and its highly variable manifestations. J. Urol. 169, 1635–1642. Napolioni, V., Curatolo, P., 2008. Genetics and molecular biology of tuberous sclerosis complex. Curr. Genomics 9, 475–487. Nelson, C.P., Sanda, M.G., 2002. Contemporary diagnosis and management of renal angiomyolipoma. J. Urol. 168, 1315–1325. Povey, S., et al., 1994. Two loci for tuberous sclerosis: one on 9q34 and one on 16p13. Ann. Hum. Genet. 58, 107–127. Qin, W., et al., 2011. Angiomyolipoma have common mutations in TSC2 but no other common genetic events. PLoS One 6, e24919. Rakowski, S.K., et al., 2006. Renal manifestations of tuberous sclerosis complex: incidence, prognosis, and predictive factors. Kidney Int. 70, 1777–1782. Roach, E.S., et al., 1998. Tuberous sclerosis complex consensus conference: revised clinical diagnostic criteria. J. Child Neurol. 13, 624–628. Shepherd, C.W., et al., 1991. Causes of death in patients with tuberous sclerosis. Mayo Clin. Proc. 66, 792–796. Tong, Y.C., et al., 1990. Renal angiomyolipoma: report of 24 cases. Br. J. Urol. 66, 585–589. van Slegtenhorst, M., et al., 1997. Identification of the tuberous sclerosis gene TSC1 on chromosome 9q34. Science 277, 805–808. Yamashita, Y., et al., 2000. Analysis of all exons of TSC1 and TSC2 genes for germline mutations in Japanese patients with tuberous sclerosis: report of 10 mutations. Am. J. Med. Genet. 90, 123–126.
Contents lists available at ScienceDirect
Experimental and Molecular Pathology journal homepage: www.elsevier.com/locate/yexmp
The analysis of mutations and exon deletions at TSC2 gene in angiomyolipomas associated with tuberous sclerosis complex Heung-Mo Yang a,1, Hye-Jung Choi a,1, Doo-Pyo Hong a, Sung-Yeon Joo a,b, Na-Eun Lee a,b, Ji-Young Song c, Yoon-La Choi d,i, Jeeyun Lee e,i, Dongil Choi f,i, BoKyung Kim g,i, Hyo-Jun Park a,h, Jae-Berm Park a,h,⁎, Sung Joo Kim a,h,i,⁎ a
Transplantation Research Center, Samsung Biomedical Research Institute, Samsung Medical Center, Seoul, Republic of Korea Samsung Advanced Institute for Health Sciences & Technology, Graduate School, Department of Health Sciences & Technology, Sungkyunkwan University, Republic of Korea c Laboratory of Cancer Genomics and Molecular Pathology, Samsung Biomedical Research Institute, Samsung Medical Center, Seoul, Republic of Korea d Department of Pathology, Samsung Medical Center, Samsung Biomedical Research Institute, Sungkyunkwan University School of Medicine, Seoul, Republic of Korea e Department of Hematological oncology, Samsung Medical Center, Samsung Biomedical Research Institute, Sungkyunkwan University School of Medicine, Seoul, Republic of Korea f Department of Radiology, Samsung Medical Center, Samsung Biomedical Research Institute, Sungkyunkwan University School of Medicine, Seoul, Republic of Korea g Department of Radiation Oncology, Samsung Medical Center, Samsung Biomedical Research Institute, Sungkyunkwan University School of Medicine, Seoul, Republic of Korea h Department of Surgery, Samsung Medical Center, Samsung Biomedical Research Institute, Sungkyunkwan University School of Medicine, Seoul, Republic of Korea i Sarcoma Research Center, Samsung Medical Center, Seoul, Republic of Korea b
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Article history: Received 2 September 2014 Accepted 12 September 2014 Available online 1 October 2014 Keywords: Angiomyolipoma Tuberous sclerosis complex TSC2 gene Mutation Exon deletion
a b s t r a c t Angiomyolipomas (AMLs) are relatively rare hamartomatous or benign tumors that occasionally occur as part of tuberous sclerosis complex (TSC). Mutations in either of the two genes, TSC1 and TSC2, have been attributed to the development of TSC. Between 1994 and January 2009, 83 patients were diagnosed with AML at the Samsung Medical Center. In that group of patients, 5 (6%) had AML with TSC (AML-TSC). Mutational analysis of the TSC2 gene was performed using 7 samples from the 5 AML-TSC patients and 14 samples from 14 patients with sporadic AML without TSC (AML-non-TSC). From this analysis, mutations in TSC genes were identified in 5 samples from the AMLTSC patients (mutation detection rate = 71%) and 3 samples from AML-non-TSC patients (mutation detection rate = 21%). In the case of AML-TSC, 6 mutations were found including 3 recurrent mutations and 3 novel mutations, while in the case of AML-non-TSC, 4 mutations were identified once, including 1 novel mutation. Also MLPA analysis of the TSC2 gene showed that TSC2 exon deletion is more frequently observed in AML-TSC patients than in AML-non-TSC patients. This is the first mutation and multiplex ligation-dependent probe amplification (MLPA) analyses of TSC2 in Korean AMLs that focus on TSC. This study provides data that are representative of the distribution of mutations and exon deletions at TSC genes in clinically diagnosed AML-TSC cases of the Korean population. © 2014 Elsevier Inc. All rights reserved.
1. Introduction Angiomyolipomas (AMLs) are relatively rare hamartomatous or benign tumor-like lesions composed of three elements: blood vessels, smooth muscles, and adipose tissues (Folpe and Kwiatkowski, 2010). Classical AML possesses the three mentioned elements but rarely contains oncocytic, cystic, or epithelioid morphologic characters; in contrast, such features are present in atypical and epithelioid AML
⁎ Corresponding authors at: Department of Surgery, Samsung Medical Center, Sungkyunkwan University School of Medicine, 50 Irwon-Dong, Gangnam-Gu, Seoul 135-710, Republic of Korea. E-mail addresses: [email protected], [email protected] (J.-B. Park), [email protected], [email protected] (S.J. Kim). 1 These authors contributed equally to this work as first and corresponding authors.
http://dx.doi.org/10.1016/j.yexmp.2014.09.013 0014-4800/© 2014 Elsevier Inc. All rights reserved.
(Belanger et al., 2005). In epithelioid AML, malignant behavior has been reported with local recurrence and distant metastasis, while classic AML is benign (Cibas et al., 2001; Kawaguchi et al., 2002). According to previous studies, AML primarily occurs in the kidney but can occur in the liver and other organs (Eble, 1998). Although the majority of AMLs are sporadic, in 20% of AML patients, they manifest with tuberous sclerosis complex (TSC); additionally, 60–80% of adult renal AML patients have TSC (Eble, 1998; Rakowski et al., 2006). TSC is an autosomal dominant disorder that is characterized by the development of multiple hamartomas in many internal organs such as the brain, heart, lung, skin, and kidney (Roach et al., 1998). Wellknown clinical manifestations of TSC include epilepsy, learning difficulties, behavioral problems, and skin lesions. The genetic basis of TSC comes from the mutation of either of the two unlinked genes, TSC1 and TSC2 (Povey et al., 1994). The human TSC1 gene is located on
H.-M. Yang et al. / Experimental and Molecular Pathology 97 (2014) 440–444
chromosome 9q34 and consists of 23 exons; the corresponding mRNA transcript is 8.6 kb, with a 3.5 kb coding region encoding a 130-kDa protein comprising 1164 amino acids (van Slegtenhorst et al., 1997). The TSC2 gene is located on chromosome 16p13.3 and contains 41 exons encoding a 200-kDa protein of 1807 amino acids (European Chromosome 16 Tuberous Sclerosis, 1993; Povey et al., 1994). The mutational spectra of the TSC genes are very heterogeneous, and no hotspots for mutations have been reported. In this study, we analyzed mutations and exon deletions of TSC2 gene in AML patients with the goal of determining the mutation in Korean individuals with both AML and TSC and providing information on the genetic character of tuberous sclerosis complex. 2. Materials and methods 2.1. Patients Between 1994 and January 2009, 83 patients were treated surgically for pathology-diagnosed AML at the Samsung Medical Center. Clinical information was collected retrospectively from patient medical records, including age at presentation, gender, involved organ, size, multiplicity, disease recurrence, and follow-up information. Clinicians assessed the general clinical features of TSC patients in accordance with the TSC diagnosis criteria set forth by the Tuberous Sclerosis Consensus Conference (Roach et al., 1998). TSC diagnosis was categorized as definite in patients with AML and other major symptoms of TSC, such as facial angiofibromas or lymphangiomyomatosis, and TSC diagnosis was categorized as probable in patients with multiple large AMLs or AML with 1 minor feature such as multiple renal cysts or liver lesions. For the purpose of mutational analysis, we divided the AML patients into two groups according to the presence (AML-TSC; n = 5) or absence (AMLnon-TSC; n = 14) of an association with TSC (Fig. 1).
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using TSC2 genomic sequence information (GenBank accession numbers AC005600.1, NM_000548.3, and NP_000539.2). PCR was performed using h-Taq DNA polymerase (Solgent, Daejeon, Korea) in 30-μl reaction volumes. Reactions were denatured at 95 °C for 20 s, annealed at 60 °C for 40 s, and extended at 72 °C for 1 min. Following the PCR reactions, we performed DNA sequencing on an ABI 3730XL DNA sequencer using a Big Dye Terminator v3.1 Cycle Sequencing Kit (Applied Biosystems, Foster City, CA, USA). 2.3. Multiplex ligation-dependent probe amplification (MLPA) analysis Genomic DNA was prepared from paraffin-embedded AML-TSC and AML-non-TSC patient tissues. Paraffin embedded human lung tissues (n = 4) were used as normal control samples. Probable identified deletions were confirmed using an MLPA kit (SALSA MLPA P046-C1 TSC2, Lot 1011; MRC-Holland, Amsterdam, The Netherlands), which contains probes for 41 TSC2 exons, according to the manufacturer's instructions. Reaction products were separated by capillary electrophoresis on an ABI 3700 sequencer (Applied Biosystems, Foster City, CA, USA) and data were analyzed using Genemarker software, version 2.5.2 (Softgenetics, State College, PA, USA). This program identifies peaks as ‘normal’ when the ratio of sample peak height or area to that of control samples falls between 0.75 and 1.30, ‘deleted’ when the ratio is b0.75, and ‘duplicated’ when the ratio is N1.30. 2.4. Statistical analysis Statistical comparisons were performed using Student's t-test with IBM SPSS (IBM Co., Armonk, NY, USA) computer software (version 12.0). Results were considered to be significant when p-values were less than 0.05.
2.2. Mutational analysis
3. Results
The mutational analysis of TSC2 genes was performed using tissue samples (n = 7) from AML-TSC patients (n = 5), including recurrent cases, and tissue samples (n = 14) from selected AML-non-TSC patients (n = 14). Genomic DNA extract kits (QIAamp® DNA FFPE Tissue Kit; QIAGEN, Valencia, CA, USA) were used according to the manufacturer's recommendations. TSC2 primers (Supplemental Table 2) were designed
3.1. Patient characteristics The clinical characteristics of patients with AML-TSC (n = 5) and AML-non-TSC (n = 14) are described in Table 1. Five of the 83 patients (6%) were AML-TSC patients; among these patients, 3 fulfilled the criteria for definitive tuberous sclerosis, and 2 were considered probable for TSC (Table 1). Comparing the clinical features between groups, patients with AML-TSC had significantly larger average AML tissue diameter(s) compared to those of the AML-non-TSC (21.4 cm vs. 5.2 cm, P b 0.001) and had multiple lesions more often than did the AML group (P = 0.004, Supplemental Table 1). Although the difference was not statistically significant, the AML-TSC group was younger and had a higher percentage of women than the AML-non-TSC group. Furthermore, we identified 3 cases of disease recurrence among all AML patients: 2 cases in the AML-TSC group and 1 case in the AMLnon-TSC group. 3.2. Mutations of TSC2 gene in AML-TSC patients
Fig. 1. Schematic diagram of TSC2 gene mutation and MLPA analysis in AML patients. For the mutation and MLPA analysis, the AML patients were divided into two groups according to the presence (AML-TSC; n = 5) or absence (AML-non-TSC; n = 14) of an association with TSC.
We performed a mutational analysis of the TSC2 coding exons in 7 samples from 5 AML-TSC patients. From the mutational analysis of the AML-TSC group samples, TSC2 gene mutations were detected in 5 samples (Patients Nos. 19A/B, 54A/B and 79), resulting in a 71% mutation detection rate (Table 1, Fig. 2A–F). As shown in Table 1, a total of 9 mutations were detected in the TSC2 genes of 3 patients from the AML-TSC group. We identified 3 unique mutations that were only detected in samples from patients with recurrent disease. Of the 6 types of mutations identified in the TSC2 gene, 1 of the 6 mutations was a nonsense mutation that resulted in an early termination codon, and the other 5 were silent mutations that altered the nucleotide sequence without affecting the TSC2 amino acid sequence (Table 1).
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Table 1 Clinical features and TSC2 gene mutation analysis of AML-TSC and AML-non-TSC patients.
3.3. Mutations in the TSC2 gene in AML-non-TSC patients From the 78 AML-non-TSC patients, we selected 14 patient samples to compare with the samples from the AML-TSC patients. Although only 1 patient with multiple tumors (a minor TSC symptom) is represented in the 14 patient samples, we could not find any other symptoms for TSC diagnosis in the 14 non-TSC patients. A mutational analysis of the TSC2 gene was performed for the 14 samples from the 14 AML-non-TSC patients. From the results of our mutational analysis, we identified 4 mutations in 3 of the 14 AML patients, representing a mutation detection rate of 21%. The 4 TSC2 mutations included 2 missense mutations, i.e., the c.1378 GNA mutation in exon 13 causing p.A460T and the c.1100 GNA mutation in exon 10 causing p.R367Q, and 2 nonsense mutations. Three of these mutations had been previously reported, while 1 mutation (c.1556_1557insG in exon 14) had not been reported (Table 1, Fig. 2G–J).
3.4. MLPA analysis of the TSC2 gene in AML-TSC and AML-non-TSC patients After mutational analysis, we analyzed exon deletion in TSC2 gene using MLPA analysis with 7 samples from 5 AML-TSC patients and 11 samples from 11 AML-non-TSC patients. According to the MLPA analysis, 4 to 8 exon deletions were identified in 7 samples of AML-TSC patients (average 5.9 deletion events/sample). Hence, in 11 samples of AMLnon-TSC patients, 0 to 5 exon deletions were identified (average 3.3 deletion events/sample; Table 1, Supplementary Fig. 1). In addition, deletions
in the TSC2 gene are commonly detected in exons 03, 04, 40 and 41 in AML patients (Supplementary Fig. 2) and TSC2 exon deletion is observed more frequently in AML-TSC patients than in AML-non-TSC patients. 4. Discussion Angiomyolipomas are benign tumors that develop mostly in the kidney and contain varying amounts of smooth muscle, fat, and blood vessels. AML is a relatively rare neoplasm that has been identified in less than 0.2% of the general population, although it is extremely common in patients with tuberous sclerosis complex (Folpe and Kwiatkowski, 2010; Lane et al., 2008). Among TSC patients, AMLs are an important diagnostic criteria in both adults and children (Jozwiak et al., 2000), and the AMLs ultimately leading to end-stage renal failure and renal disease as causes of death (Shepherd et al., 1991). Moreover, mutations in the TSC1 and TSC2 genes have been attributed to the development of TSC, and mutational analysis is used in the diagnosis of TSC. In this study, we reported the clinical features of AML patients and firstly performed a mutational analysis of the coding exons of the TSC2 gene to investigate the association of mutations with TSC in Korean populations. TSC2, rather than TSC1, was studied because TSC1 gene mutations are more rare than TSC2 gene mutations (Qin et al., 2011) and because we were unable to detect TSC1 mutations in 7 samples from 5 AML-TSC patients (data not shown). Our study demonstrated a relatively low prevalence rate for AML associated with TSC (5 of 83 patients, 6%) compared with the rate of 20%
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Fig. 2. Representative sequencing traces demonstrating TSC2 mutations. A–F. Mutations in the TSC2 genes of AML-TSC patients. G–J. Mutations in the TSC2 genes of AML-non-TSC patients. Red arrows indicate the point of mutation.
reported by Lendvay and Marshall (2003). However, our identified rate is consistent with previous studies finding that fewer than 10% of all AMLs occur in individuals with TSC (Aydin et al., 2009; Chen et al., 1997; Tong et al., 1990). According to previous reports, AMLs associated with TSC were found in significantly younger patients, had a larger mean tumor size, and had more lesions than did those without TSC (Aydin et al., 2009; Nelson and Sanda, 2002). Additionally, AML is approximately twice as frequent in women as in men, both in the general population and in TSC patients (Shepherd et al., 1991; Tong et al., 1990). Similarly, our study demonstrated that AML-TSC patients had significantly larger AMLs, multiple lesions, and frequent disease recurrences (Table 1, Supplemental Table 1). In TSC patients, mutations in the TSC1 and TSC2 genes are detected in a maximum of 80–90% of patients, with only a single relatively small series achieving a mutation detection rate of 91% (Jansen et al., 2008). In this study, we identified 5 mutations in 7 samples from the AML-TSC group, corresponding to a mutation detection rate of 71%, and all of these mutations were in the TSC2 gene. The mutation rate observed in our samples is lower than that of other western countries and Japan (Dabora et al., 2001; Jones et al., 1997, 1999; Yamashita et al., 2000). Moreover, many reports have suggested that mutations in TSC2 are about five times more common in the sporadic TSC population than mutations in TSC1. Although the incidence of mutations in our study is slightly lower than in previous reports, we observed a lower mutation rate in the TSC2 gene (21%) compared to that found for the AML-TSC group (Table 1). To date, over 1500 different mutations in TSC1 and TSC2 have been reported (Cheadle et al., 2000). Among these mutations, deletions, nonsense, and missense mutations occur at nearly equal frequencies in TSC2 (22–27%). According to a previous study, most lesions are distributed over the entire TSC2 gene region and include point mutations leading to nonsense mutations, small deletions and insertions, splice site changes, and missense mutations (Napolioni and Curatolo, 2008). In our MLPA assay, we observed several deleted and amplified TSC2 exons
(Supplemental Fig. 1). TSC2 exon deletion was observed more frequently in the samples of AML-TSC patients (average 5.9 events) than in the samples of AML-non-TSC patients (average 3.3 events). In addition, not only mutation presence and deletion presence in a specific gene are important, but also mutation status and genomic rearrangement are necessary factors to understand genetic alteration. Therefore, the patterns of the gene inactivation (one/two copy gene inactivations) and genomic event (inversion, frame-shift, insertion) are valuable information. We also know that LOH analysis is needed for the analysis of genomic alteration as well as for the inactivation pattern of a specific gene. However, unfortunately, it is difficult to perform LOH analysis, because we have no normal part of tissue sample from patients. Based on our analysis, it seems that a relatively high rate of mutation and deletion events in the TSC2 gene might be of concern for TSC progression and diagnosis. In our results, there are two pairs of samples from the same patients (patient nos. 19A/B and 54A/B). It seems that the mutations occurred independently, with the exception of mutation of c.228 CNT in exon 03. This mutation has a possibility of being a germline mutation because it was detected in both samples from one patient (patient no. 54A/B); however, we cannot make any assertions in this matter. In conclusion, our study is the first analysis identifying TSC2 gene mutations and deletions from AML-TSC patients in Korea. Among 83 AML patients, 5 patients were designated as having AML-TSC. The results of our mutational analysis of TSC2 genes in AML-TSC show that six different mutations were identified (a mutation detection rate of 71%), while 4 different mutations were identified in AML-non-TSC (a mutation detection rate of 21%). Moreover, TSC2 exon deletion is observed more frequently in AML-TSC patients than in AML-non-TSC patients. Although the incidence of TSC mutations is slightly lower than that found in previous mutational analyses, TSC mutation-related clinical features of AML-TSC patients were similar to previous reports. Moreover, for recurrent AML patients, repeated mutations of TSC2 were observed in multiple unrelated patients and in multiple samples from
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the same patient. The identification of additional TSC2 gene mutations and exon deletions will help confirm a TSC diagnosis and will aid in the clinical management of AML-TSC patients. Supplementary data to this article can be found online at http://dx. doi.org/10.1016/j.yexmp.2014.09.013. Conflict of interest statement No potential conflicts of interest were disclosed. Acknowledgment This research was supported by Samsung Biomedical Research Institute grant, SBRI C-B1-105-3 and supported by Basic Science Research Program through the National Research Foundation of Korea (NRF) funded by the Ministry of Education (NRF-2013R1A1A2063324). References Aydin, H., et al., 2009. Renal angiomyolipoma: clinicopathologic study of 194 cases with emphasis on the epithelioid histology and tuberous sclerosis association. Am. J. Surg. Pathol. 33, 289–297. Belanger, E.C., et al., 2005. Epithelioid angiomyolipoma of the kidney mimicking renal sarcoma. Histopathology 47, 433–435. Cheadle, J.P., et al., 2000. Molecular genetic advances in tuberous sclerosis. Hum. Genet. 107, 97–114. Chen, S.S., et al., 1997. Renal angiomyolipoma—experience of 20 years in Taiwan. Eur. Urol. 32, 175–178. Cibas, E.S., et al., 2001. Malignant epithelioid angiomyolipoma (‘sarcoma ex angiomyolipoma’) of the kidney: a case report and review of the literature. Am. J. Surg. Pathol. 25, 121–126. Dabora, S.L., et al., 2001. Mutational analysis in a cohort of 224 tuberous sclerosis patients indicates increased severity of TSC2, compared with TSC1, disease in multiple organs. Am. J. Hum. Genet. 68, 64–80. Eble, J.N., 1998. Angiomyolipoma of kidney. Semin. Diagn. Pathol. 15, 21–40. European Chromosome 16 Tuberous Sclerosis, C., 1993. Identification and characterization of the tuberous sclerosis gene on chromosome 16. Cell 75, 1305–1315.
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